Nephrogenic diabetes insipidus (NDI) can be caused by hypercalcemia. The effect of early onset NDI in parathyroid hormone (PTH)-induced hypercalcemia was studied using proteomics and phosphoproteomics profiling of rat kidney medulllary collecting duct (IMCD). Tryptic peptides and phosphopeptides were identified and quantified by mass spectrometry using a label-free methodology. A total of 5,866 peptides corresponding to 1,107 proteins and 1,388 phosphopeptides of 580 proteins were identified, with significant changes in abundance of 69 proteins and 49 phosphopeptides in early onset NDI. Gene Ontology terms and pathway analysis revealed that hypercalcemia-affected proteins and phosphoproteins are from integrin signaling pathway and actin cytoskeleton organization. Immunoblotting revealed altered abundances of vasopressin-regulated phosphorylation of aquaporin-2 (Aqp2) at Ser256 and Ser261, and decreased abundance of urea transporter UT-A1 (Slc14a2) and UT-A3, together with their phosphorylation at Ser486. This is the first report of the involvement of integrin signaling pathway in urine concentration mechanism in hypercalcemia-induced early onset NDI.